Polymeric carrier for a controlled synthesis of peptides

ABSTRACT

A new acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides is provided. Such a polymer comprises the reaction product of (1) a hydroxyalkyl or aminoalkyl acrylate or methacrylate or an acrylanide or methacrylanide, (2) an alkylene diacrylate or dimethacrylate or an acrylate or methacrylate of a polyalcohol, a dihydroxy or polyhydroxy ether, or an alcoholic sugar or an alkylene bisacrylamide or divinylbenzene, and (3) acrylanilide or metacrylanilide or phenyl acrylate or methacrylate or a phenoxyalkyl acrylate or methacrylate or styrene, the aromatic ring being chloromethylated.

United States Patent Coupek et al.

[ POLYMERIC CARRIER FOR A CONTROLLED SYNTHESIS OF PEPTIDES [75] Inventors: .liri Cou pek, Prague; Vladimir Gut, Uhrineves, both of Czechoslovakia [73] Assignee: Ceskoslovenska akademie ved,

Prague, Czechoslovakia 22 Filed: Nov. 6, 1973 21 Appl. No.:413,390

[30] Foreign Application Priority Data Nov 6, l972 Czechoslovakia 7475-72 [52] US. Cl. 260/25 R; 260/2.5 l-lB; 260/6; 260/l7.4 SB; 260/47 UA; 260/52; 260/72; 260/725; 260/73 L; 260/8073; 260180.81

[5]] Int. CL? C08J 9/00; C08G 12/08; C08G 12/46 [58] Field of Search 260/67 A, 73 L, 80.73,

[ Dec. 9, 1975 Primary Examiner-Morton Foelak Attorney, Agent, or FirmMurray Schaffer [57] ABSTRACT A new acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides is provided. Such a polymer comprises the reaction product of l) a hydroxyalkyl or aminoalkyl acrylate or methacrylate or an acrylanide or methacrylanide, (2) an alkylene 6 Claims, No Drawings POLYMERIC CARRIER FOR A CONTROLLED SYNTHESIS OF PEPTIDES The invention relates to a new polymeric carrier, the method of its preparation and its application in a controlled synthesis of polypeptides.

Biologically active polypeptides may be formed by a stepwise condensation of definite aminoacids in the precisely defined sequence. The classic preparation of such compounds by condensation in solution requires the pretentious isolation of reaction products after every reaction step and their characterization. However, this procedure is experimentally unfeasible for peptides with longer chains. Merrifield (P. B. Merrifield; J. Am. Chem. Soc., 85, 2149 (1963)) described a method overcoming the complications inhering in sequential stepwise condensation of aminoacids. This method consists of three states linking of the first amonoacid to a solid polymer carrier by covalent bond, sequential stepwise reaction of further aminoacids to form the peptide chain, and the eventual cleavage of the formed chain from the carrier and its isolation without damage.

If the growing peptide chain is covalently bound to the solid carrier during the whole sequence of stepwise condensation reactions, separation of products from unreacted agents and products of side reactions is very facilitated in each reaction step. Complicated isolation operations, which are necessary in the solution process, are reduced to elution or decanting of particles of the solid carrier with anchored growing polypeptide. This procedure simplified the synthesis significantly, considerably shortened the time of synthesis and made its automation possible.

A highly crosslinked copolymer of styrene with divinylbenzene, which is activated for reaction with the first aminoacid, is usually used as the carrier for polypeptide synthesis. This material has two substantial disadvantages limiting its general application. This is, in the first place, the physical structure of the copolymer, which is a xerogel and exhibits therefore a considerable dependence of mechanical properties on the polymer network density. The condensation reactions of aminoacids take place inside the physically homogeneous gel matter and the network density forms a natural limit for the molecular weight synthetized polypeptide. Diffusion coefficients of reaction components, which have to be brought to the reaction site at the growing polypeptide chain, as well as those of reaction products, which have to be washed out after completion of the reaction, are substantially lower in the homogeneous xerogel than in solution. This fact may cause slower establishing of equilibrium in condensation reactions, negatively influence the final degree of conversion in individual reaction steps and, eventually, render more difficult and longer washing of the material after the elementary condensation reaction. Another disadvantage of the styrene gel follows also from its homogeneously crosslinked structure; namely, it can be used only in solvents swelling the copolymer. Because these are exclusively organic solvents often of low polarity, the choice of reaction possibilities for aminoacid condensation is automatically limited. Application of aqueous reaction systems is impossible. Sorption interactions of some aminoacids with the nonpolar gel carrier may be expected in the non-aqueous medium, which fact can further render their removing after completion of the condensation step more difficult. All these facts result in non-quantitative course of the individual reaction steps in linking aminoacids and, consequently, some aminoacids are missing in the resulting reaction product after its splitting from the carrier. This can 5 very unfavourably affect the biological efficiency of the product.

The polymeric carrier according to th s invention consists of a hydrophilic gel, which contains besides hydrophilic functionalgroups also aromatic rings and wherein the primary carrier is further activated by amides or methacrylamides linked with a crosslinking monomer of acrylate or methacrylate type and with the third or even further monomer containing aromatic rings in its molecule, which can be activated by chloromethylation. The crosslinking comonomers of acrylate or methacrylate type may be alkylene diacrylates, al-

kylene dimethacrylates, polyacrylates or polymethacrylates of polyalcohols, dihydroxyor polyhydroxyethers or alcoholic sugars, alkylene bis-acrylamides or divinylbenzene. The object of this invention is further a method for preparation of the gel carrier, which does not possess the negative properties of polystyrene homogeneous gels generally used till now, and application of this gel in controlled synthesis of polypeptides. The carriers are prepared, according to this invention, by a suspension heterogeneous copolymerization of hydrophilic acrylate or methacrylate monomers with the crosslinking comonomer in the presence of a third comonomer containing aromatic ring in the molecule, as for example phenly acrylate, phenyl methacrylate,

acrylanilidc, methacrylanilide, phenoxyalkyl acrylate or methacrylate, styrene and others. Materials with similar properties may be formed also in a subsequent modification of macroporous suspension copolymers prepared according to Czechoslovak Patent no.

150,819 corresponding to US. patent application Ser.

No. 281,288, by compounds containing the aromatic ring and forming condensation compounds with the pendent hydroxyl group of the polymer, as for example by aromatic acids, their chlorides or anhydrides, aromatic isocyanates, halo genophenylsilanes, or aromatic epoxy compounds. Finally, the primary carriers can be prepared by polymerization reactions followed by diazotization of the resulting polymers and coupling with a passive aromatic component. All these copolymers have to be able to undergo chloromethylation to be activated for the reaction with the first aminoacid.

The gels with macroporous structure are formed in the process of heterogeneous suspension copolymerization and are noted for the high mechanical strength and hydrolytic stability. Their specific surface area ranges between 5 and 500 in per gramm of the copolymer and they may be prepared in a form of globular particles of uniform size. During the activation, only a part of aromatic rings reacts, which is exposed to action of formal dehyde and hydrogen chloride at the surface of copolymer. When the material has large specific surface area, also its capacity is high enough in comparison with homogeneous materials. Then the synthesis of peptide takes place on the solid surface of the copolymer,

55 which does not swell or swells only slightly due to its properties of aerogel and therefore allows to use arbitrary polar or nonpolar organic solvent as the reaction medium. All complications connected with the slow es- 3 tablishing of reaction equilibria and transport of products from the reaction site, both controlled by diffusion in the gel, are also taken away.

The object of the invention is illustrated in the following examples, without, however, limiting the scope of the invention.

EXAMPLE 1 A macroporous hydrophilic copolymer prepared by the heterogeneous suspension copolymerization of 20 weight parts of methacrylanilide, 40 wt. parts of 2- hydroxyethyl methacrylate and 40 wt. parts of ethylene dimethacrylate in the presence of inert solvents according to Czechoslovak Pat. No. 150,819 (laurylalcohol, cyclohexanol) was used as the primary carrier and activated by chloromethylation. This gel (2 wt. parts), 24 wt. parts of zinc (ll) chloride and 12 wt. parts of concentrated hydrochloric acid were mixed with 10 wt. parts of aqueous formaldehyde solution (38%). The suspension was heated to 50C and a stream of gaseous hydrogen chloride was led through it for 5 hours. The gel was then filtered, washed with 100 wt. parts of water and 100 wt. parts of methanol and dried in vacuum over P and NaOH for 12 hours. An analytical determination of chlorine showned 5.92% CI.

The copolymer activated by chloromethylation (2 wt. parts) was suspended in a solution of 0.456 wt. part of o-nitrobenzenesulphenylglycine and 0.25 wt. parts of triethylamine in 8 wt. parts of dimethylformamide and stirred at 25C for 120 hours. The suspension was then diluted with 50 wt. parts of dimethylformamide and the gel was decanted, washed gradually with methanol, water, methanol, and dichloromethane and then dried in vacuum. After hydrolysis, 0.25 mmole of glycine per gramm of the gel was determined by an aminoacid analysis.

One wt. part of the gel with bound o-nitrobenzenesulphenylglycine was washed with 50 wt. parts of 0.1 M HCL in the methanol dichloromethane mixture (1 l) on a glass filter during 15 min and then with pure dichloromethane. The quantitative cleavage of the protecting o-nitrobenzenesulphenyl groups (0.25 mmole/g) was proved by measuring the optical density of filtrate at 384 nm. The aminoacid was released from its salt by elution with a solution of 1.2 wt. parts of triethylamine in 10 wt. parts of dichloromethane and the gel was then washed with pure dichloromethane. The gel was then suspended in 10 wt. parts of dichloromethane, 0.12 wt. part of o-nitrobenzenesulphenylalanine and 0.105 wt. part of dicyclohexylcarbodiimide was added to the suspension and the mixture was stirred at 25C for 30 minutes. The gel was filtrated, washed with dichloromethane, dried in vacuum over P 0 and analyzed on the content of bound aminoacids.

Splitting of the peptide from the polymeric carrier was carried out by hydrogen bromide in trifluoroacetic acid. The peptidyl copolymer (0.2 wt. part) was mixed into 3 wt. parts of anhydrous trifluoroacetic acid and dry gaseous hydrogen bromide was led in at 0C for 1 hr. After evaporation, the splitted product was washed from the carrier with ml of water and refined by chromatography.

EXAMPLE 2 The synthesis was carried out similarly as in Example 1, with the distinction that 0.175 wt. part of tertbutyloxycarbonylglycine was used for bonding instead of o-nitrobenzenesulphenylglycine. After hydrolysis,

0.24 mmole of glycine was found in 1 g of the copolymer by analysis on aminoacids. Tert-butyloxycarbonylglycly copolymer (0.5 wt. part) was suspended in 5 wt. parts of trifluoroacetic acid and allowed to stand at the room temperature for 30 min. The polymer was filtered off and perfectly washed with dichloromethane as far as all trifluoroacetic acid was washed out.

EXAMPLE 3 The synthesis was carried out analogously as in Ex ample l, with the distinction that N-hydroxysuccinimide method was used instead of the carbodiimide method to bind further amino-acid. N-hydroxysuccinimide ester of o-nitrobenzenesulphenylalanine (0.17 wt. part) was added into a suspension of 1 wt. part of glycyl copolymer in 10 wt. parts of dichloromethane and the reaction mixture was stirred for 1 hour at the laboratory temperature. The polymer with linked protected dipeptide was isolated by filtration, washed threetimes by 20 wt. parts of dichloromethane on the filter, sucked off and dried in vacuum over P 0 and NaOH.

EXAMPLE 4 The synthesis was carried out analogously as in Example l, with the distinction that liquid hydrogen fluoride was used for splitting the peptide from the polymeric carrier. Hydrogen fluoride was distilled in a Daiflon apparatus to 0.2 wt. part of peptidyl copolymer and the suspension was stirred at 0C for 30 min. l-lydrogen fluoride was removed by distillation and the product was washed out from the carrier with 20 wt. parts of water and refined similarly as in Example 1.

EXAMPLE 5 The synthesis was carried out analogously as in Example 1, with the distinction that peptide was splitted from the polymeric carrier by methanolic ammonium in a form of amide. The peptidyl copolymer (0.2 wt. part) was suspended in 10 wt. parts of methanol, which was previously saturated with gaseous ammonium at 0C, and the suspension was allowed to stand for 48 hours at 25C being stirred occasionally. Peptide amide with protected aminogroup was obtained in the filtrate after filtration and washing with methanol and was further worked out according to the type of its protective group.

EXAMPLE 6 The synthesis was carried out similarly as in Example 1, with the distinction that peptide was splitted off by liquid ammonium in a form of amide. Dry ammonium (10 wt. parts) was distilled to 0.2 wt. part of the peptidyl copolymer in a steel autoclave. The mixture was heated to the laboratory temperature and allowed to stand for 44 hours. The autoclave was then cooled to 40C again, opened, ammonium was evaporated and the product was obtained by extraction with methanol.

EXAMPLE 7 The synthesis was carried out analogously as in Example 3, with the distinction that a macroporous copolymer of 2-hydroxyethyl methacrylate (50%), ethylene dimethacrylate (40%) and styrene 10%) was used as the primary carrier.

EXAMPLE 8 The carrier was synthetized analogously as in Example 1, with the distinction that 40 wt. parts of Z-hydroxyethyl acrylate and 40 wt. parts of ethylene diacrylate together with 20 wt. parts of phenyl acrylate were used in copolymerization.

EXAMPLE 9 The carrier was synthetized similarly as in Example 3 with the distinction that methacrylamide, methylenebis-acrylamide and phenoxyethyl methacrylate were used as monomers.

EXAMPLE 10 The carrier was prepared similarly as in Example 9, with the distinction that acrylamide was used instead of methacrylamide.

EXAMPLE 1 l The polymeric carrier was prepared by copolymerization of Z-aminoethyl methacrylate with methylenebis-acrylamide and phenoxyalkyl methacrylate.

EXAMPLE 12 The carrier was synthetized analogously as in Example ll, with the distinction that aminoethyl acrylate was used instead of aminoethyl methacrylate.

EXAMPLE l3 The carrier was prepared analogously as in Example 9, with the distinction that N-methylmethacrylamide, tetraethylene glycol dimethacrylate and acrylanilide were used as monomers.

EXAMPLE 14 The carrier was synthetized similarly as in Example I with the distinction that methacroylated pentaerythrito] was used as the crosslinking monomer.

EXAMPLE 15 The carrier was synthetized analogously as in Example 14, with the distinction that divinylbenzene was used as the crosslinking monomer.

EXAMPLE 16 The polymeric carrier was prepared by copolymerization of 60 wt. parts of Z-hydroxyethyl methacrylate with 40 wt. parts of ethylene dimethacrylate. The copolymer was washed and thoroughly dried and then its free hydroxyl groups were condensed with benzoyl chloride 10 wt. parts of the copolymer was treated with 25 wt. parts of benzoyl chloride. The benzoylated copolymer was washed, dried, and then activated by chloromethylation.

EXAMPLE 17 The polymeric carrier from Example l6 (10 wt. parts) was treated with 5 wt. parts of phenylisocyanate in wt. parts of benzene at 40C. The reaction product was washed, dried, and activated by chloromethylation.

EXAMPLE 18 The polymeric carrier according to Example 16 was treated with diphenyldichlorosilane in benzene solution at 40C. The weight ratio of reacting components was 6 analogous as in Example 17. The washed and dried co polymer was activated by chloromethylation.

EXAMPLE l9 5 The copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate was prepared analogously as in Example 16. p-Acetaminophenoxyethyl methacrylate (5 wt. parts) was used as the third monomer. The copolymer was hydrolyzed in 1% aqueous NaOH at 100C, washed, and diazotized by treatment with 0.1 N NaNO: and 0.1 N HCL solution. The resulting diazotized gel was coupled with 10 wt. parts of B-naphthol and then activated by chloromethylation similarly as in Example I.

We claim:

1. A polymeric gel carrier, for use in the controlled synthesis of polypeptides comprising a terpolymer of a first monomer selected from the group consisting of hydroxyalkyl acrylates, hydroxyalkyl methacrylates, aminoalkyl acrylates, aminoalkyl methacrylates, acrylamides and methacrylamides, a polyfunctional crosslinked monomer of acrylate or methacrylate, and a third monomer which contains an aromatic ring selected from the group consisting of acrylanilide, methacrylanilide, phenyl acrylate, phenyl methacrylate, phenoxyalkyl acrylates, phenoxyalkyl methacrylates, and styrene, said aromatic rings being chloromethylated under the action of formaldehyde and hydrogen chloride.

2. A polymeric carrier, according to claim 1 wherein the crosslinking monomer is selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, polyacrylates and polymethacrylates of polyalcohols, alkylene bisacrylamides and divinylbenzene.

3. A polymeric carrier according to claim 1, wherein the polymer gel has a macroporous structure.

4. An acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides, which comprises the reaction product of a. a monomer selected from the group consisting of hydroxyalkyl acrylates, hydroxyalkyl methacry lates, aminoalkyl acrylates, aminoalkyl methacrylates, acrylamides, and methacrylamides;

b. a monomer selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, polyacrylates and polymethacrylates of polyalcohols, alkylene bisacrylamides, and divinylbenzene.; and

c. an aromatic ring-containing monomer selected from the group consisting of acrylanilide, methacrylanilide, phenyl acrylate, phenyl methacrylate, phenoxyalkyl acrylates, phenoxyalkyl methacrylates, and styrene; the aromatic ring moiety being chloromethylated.

5. An acrylic polymer according to claim 4, which has a macroporous structure.

6. An acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides, which com- 60 prises the reaction product of 2hydroxyethyl methacrylate, ethylene dimethacrylate, and methacrylanilide, 

1. A POLYMERIC GEL CARRIER, FOR USE IN THE CONTROLLED SYNTHESIS OF POLYPEPTIDES COMPRISING A TERPOLYMER OF A FIRST MONOMER SELECTED FROM THE GROUP CONSISTING OF HYDROXYALKYL ACRYLATES, HYDROXYALKYL METHACRYLATES, AMINOALKYL ACRYLATES, AMINOALKYL METHYACRYLATES, ACRYLAMIDES AND METHACRYLATE, AND TIONAL CROSS-LINKED MONOMER OF ACRYLATE OR METHACRYLATE, AND A THIRD MONOMER WHICH CONTAINS AN AROMATIC RING SELECTED FROM THE GROUP CONSISTING OF ACRYLANILIDE, METHACRYLANILIDE, PHENYLACRYLATE, PHENYL METHACRYLATE, PHENOXYALKYL ACRYLATES, PHENOXYALKYL METHACRYLATES, AND STYRENE, SAID AROMATIC RINGS BEING CHLOROMETHYLATED UNDER THE ACTION OF FORMALDEHYDE AND HYDROGEN CHLORIDE.
 2. A polymeric carrier, according to claim 1 wherein the crosslinking monomer is selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, polyacrylates and polymethacrylates of polyalcohols, alkylene bisacrylamides and divinylbenzene.
 3. A polymeric carrier according to claim 1, wherein the polymer gel has a macroporous structure.
 4. An acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides, which comprises the reaction product of a. a monomer selected from the group consisting of hydroxyalkyl acrylates, hydroxyalkyl methacrylates, aminoalkyl acrylates, aminoalkyl methacrylates, acrylamides, and methacrylamides; b. a monomer selected from the group consisting of alkylene diacrylates, alkylene dimethacrylates, polyacrylates and polymethacrylates of polyalcohols, alkylene bisacrylamides, and divinylbenzene; and c. an aromatic ring-containing monomer selected from the group consisting of acrylanilide, methacrylanilide, phenyl acrylate, phenyl methacrylate, phenoxyalkyl acrylates, phenoxyalkyl methacrylates, and styrene; the arOmatic ring moiety being chloromethylated.
 5. An acrylic polymer according to claim 4, which has a macroporous structure.
 6. An acrylic polymer suitable for use as a carrier in the controlled synthesis of polypeptides, which comprises the reaction product of 2-hydroxyethyl methacrylate, ethylene dimethacrylate, and methacrylanilide, the anilide moiety being chloromethylated. 